This invention relates to a process for rapid, easy, and high-precision measurement of trace components in samples derived from living bodies, for example, body fluids such as serum, blood, plasma and urine, lymphocyte, hemocyte, and various cells.
It is known that specific substances interact strongly on each other (namely, they have a high affinity for each other) to form a firm complex. The specific substances include, for example, the following combinations: antigen and antibody; protease and its protein protease inhibitor; sugar chain and lectin; enzyme and substrate therefor or coenzyme; physiologically active substance such as hormone, and receptor or transport protein for said active substance; and a pair of polynucleotide chains of duplex DNA. There are widely employed methods in which trace components in samples are purified or assayed by utilizing the above interaction.
Typical examples of methods for measuring a trace component in a sample by utilizing the above interaction include a method in which an equilibrium state which results from the intereaction between an analyte to be measured and a substance having affinity for the analyte (hereinafter abbreviated as "affinity substance") is measured using a labeling substance, whereby the trace component is measured. More specific examples include radioimmunoassay (RIA), enzyme immunoassay (EIA) and fluoroimmunoassay (FIA).
In more detail, the methods for measuring trace components by measuring the equilibrium state which results from the interaction are roughly divided into the following two categories: so-called noncompetitive reaction methods which comprise reacting an affinity substance labeled with a labeling substance (hereinafter abbreviated as "labeled affinity substance") with an analyte to be measured, determining the amount of the labeling substance in the resulting complex of the analyte and the affinity substance (hereinafter abbreviated as "complex"), and thereby determining the amount of the analyte; and so-called competitive reaction methods which comprise reacting an analyte to be measured with the analyte which has been labeled with a labeling substance (hereinafter abbreviated as "labeled analyte") and an affinity substance, determining the amount of the labeling substance in the resulting complex of the labeled analyte and the affinity substance (hereinafter abbreviated as "labeled complex"), and thereby determining the amount of the analyte. The methods of each category can be further divided into so-called homogeneous methods in which measurement is carried out without separating the complex formed by the interaction from free (uncomplexed) labeled affinity substance (or labeled analyte), and so-called heterogeneous methods in which measurement is carried out after separating the complex formed by the interaction with free (uncomplexed) labeled affinity substance (or labeled analyte).
In the homogeneous method, the equilibrium state which results from the interaction is measured by utilizing the phenomenon that a labeling substance is activated (or inactivated) by the formation of the complex. Therefore, measuring procedure and the like in this method are simple, but this method is disadvantageous, for example, in that the kinds of usable labeling substance and analyte to be measured are limited. Accordingly, this method is not widely used.
In the heterogeneous method, various labeling substances can be used and substances in wide variety can be measured. Therefore, this method is a leading method for measuring a trace component. In this method, needless to say, a procedure of separating the complex (Bound form) formed by the interaction from free (uncomplexed) labeled affinity substance (or labeled analyte) (Free form), i.e., a procedure of the so-called B/F separation, is indispensable.
The B/F separation is carried out, for example, by combining the complex with an antibody against either the analyte or affinity substance constituting the complex, which has been immobilized on an insoluble carrier, and then separating the complex combined with the antibody, together with the insoluble carrier, or by adding said antibody to the reaction solution to form a new complex with said complex, and separating the thus formed complex as precipitate. Therefore, the heterogeneous method is disadvantageous, for example, in that it requires a troublesome procedure, requires a long time before measurement, and does not permit easy automation of measurement. Accordingly, its improvement is desirable.
On the other hand, in order to solve the above problems in the heterogeneous method, there have been devised methods in which the B/F separation is carried out by a so-called affinity chromatography which uses a column packed with a carrier on which an analyte to be measured or an affinity substance for the analyte has been immobilized (Clinical Chemistry, 30,417-420 (1984); Clinical Chemistry, 30, 1494-1498 (1984); etc.)
However, in these methods, free (uncomplexed) labeled affinity substance (or labeled analyte) is removed using an affinity chromatography column having the analyte (or the affinity substance) immobilized thereon. Therefore, these methods involve, for example, the following problems; the analyte (or the affinity substance) should be previously prepared (obtained) in a relatively large amount; a packing for the affinity chromatography column should be prepared; an affinity chromatography column suitable for each analyte to be measured is needed; and said column should be regenerated when a large number of samples are dealt with. Accordingly, said methods are not always satisfactory enough.